In women with WHO type II anovulatory infertility such as polycystic ovary syndrome (PCOS), the treatment of first choice for the induction of ovulation is an anti-estrogen. The most commonly used drug is clomiphene citrate (CC). However, 20 to 25 percent of women do not ovulate with CC. In addition, clinical data reveal a discrepancy between ovulation and conception rates during CC treatment1, and a higher than expected incidence of miscarriage in conception cycles2. These observations have been attributed to the anti-estrogenic mechanism of action of CC resulting in long-lasting estrogen receptor depletion. Thus, CC may have a negative effect on the quality and quantity of cervical mucus3, on endometrial development4, and on other as yet undetermined fertility factors since CC is accumulated in the body as a result of its long half-life5.
In CC failures, gonadotropin preparations such as human menopausal gonadotropin (hMG) or pure follicle stimulating hormone (FSH) have been used as a second-line treatment for ovulation induction. In women with polycystic ovary syndrome, because of the high sensitivity of the ovaries to gonadotropin stimulation, treatment with hMG or pure FSH is difficult to control and characteristically induces multiple follicles. The result is a high frequency of multiple pregnancies and increased risk of ovarian hyperstimulation syndrome (OHSS)6. Therefore, a simple oral treatment that could be used without risk of hyperstimulation and with minimal monitoring is the preferred first line of therapy.
Additionally, although it has been established that pregnancy rates for women who take CC are less than expected based on ovulation rates, CC therapy is widely administered to women with unexplained infertility, often without ultrasound monitoring, in order to induce the development of multiple follicles7. The use of CC in these women may be unsuccessful because of antiestrogenic effects on endometrial development. A recent study has prospectively applied morphometric analysis of the endometrium, which is a quantitative and objective technique to study the effect of CC on the endometrium in a group of normal women. In this study, CC was found to have a deleterious effect on the endometrium, demonstrated by a reduction in glandular density and an increase in the number of vacuolated cells8. In some exceptional cases, normal ovulatory women may receive 6 to 12 cycles of CC before it is finally determined that the anti-estrogenic effects of CC on the endometrium are actually causing an anti-conception action. For these reasons, a simple, inexpensive and safe alternative to CC for use in normally ovulatory women, in whom frequent cycle monitoring is difficult, is also required.
The induction of ovulation constitutes a vital part of infertility management. Unfortunately, most current therapeutic approaches for induction of ovulation have been empiric9. For over 40 years, clomiphene citrate (CC) has been the most commonly used treatment for the induction and augmentation of ovulation, accounting for about two thirds of the fertility drugs prescribed in the United States10. However, the mechanism(s) and site(s) of CC action have only been partially clarified despite extensive clinical research11.
Mechanism of CC Action
It is believed that the 2 isomers of CC exert either an anti-estrogenic effect (zu-clomiphene) or a weak estrogen agonist effect (en-clomiphene) at estrogen binding sites in the pituitary and hypothalamus, thus releasing the hypothalamic/pituitary axis from the inhibitory effect of the major circulating estrogen, estradiol (E2)12. In women with PCOS, CC-induced ovulation was accompanied by increased secretion of LH and FSH with enhanced estrogen secretion. Increased LH pulse amplitude after CC, together with decreased pituitary sensitivity to a GnRH bolus, suggested that CC acted predominantly on the hypothalamus to cause release of larger pulses of GnRH into the pituitary-portal system13. Similar findings have been reported in normal ovulatory women14. Various mechanisms of CC action have also been suggested at the level of the pituitary and/or the ovary. In particular, the ovarian actions of CC have not been widely appreciated15. However, the overall mechanism of CC action may be the sum of its effects on the hypothalamus, pituitary and ovary as discussed by Adashl16.
Approaches to Improve Pregnancy Outcome with CC
In order to improve the outcome of CC treatment, various approaches have been suggested to overcome the antiestrogenic effect of CC. One approach has been to administer estrogen concomitantly during CC treatment to attain high estrogen levels to overcome the antiestrogenic effect of CC. Some investigators have reported success with this approach17 while others have reported no benefit18 or even a deleterious effect of estrogen administration19. Another approach to reduce adverse effects has been to administer CC earlier during the menstrual cycle rather than starting on day 520, in the hopes of allowing the anti-estrogenic effect to wear off to some extent. A third approach has been to combine another selective estrogen receptor modulator like tamoxifen, which has more estrogen agonistic effect on the endometrium with CC21. However, none of the above mentioned strategies have proved to be completely successful in avoiding the peripheral antiestrogenic effects of CC. In addition to a discrepancy between ovulation and pregnancy rates with CC treatment, 20% to 25% of anovulatory women are resistant to CC and fail to ovulate at doses up to 150 mg daily. In CC failures, gonadotropins have been used as a second-line treatment for ovulation induction. However, they are associated with higher risk of multiple pregnancy, and severe ovarian hyperstimulation syndrome, especially in women with PCOS. Therefore, a simple oral alternative to CC that could be used without high risk and which requires minimal monitoring would be the preferred first line of therapy for ovulation induction.
Aromatase Inhibitor
A group of highly selective AIs has been approved for use in postmenopausal women with breast cancer to suppress estrogen production. These AIs have a relatively short half-life (approximately 48 hours) compared to CC, and therefore would be eliminated from the body rapidly22. In addition, since no estrogen receptor down-regulation occurs, no adverse effects on estrogen target tissues, as observed in CC treated cycles, would be expected.
Physiology of Aromatase Enzyme
Aromatase is a cytochrome P-450 hemoprotein-containing enzyme complex that catalyzes the rate-limiting step in the production of estrogens, i.e. the conversion of androstenedione and testosterone, via three hydroxylation steps, to estrone and estradiol23. Aromatase activity is present in many tissues, such as the ovaries, adipose tissue, muscle, liver, breast tissue, and in malignant breast tumors. The main sources of circulating estrogens are the ovaries in premenopausal women and adipose tissue in post-menopausal women24. Although aromatase has features in common with other steroidogenic P-450 enzymes, the heme-binding region has only 17.9±23.5% amino acids identical to those of other steroidogenic P-450 enzymes. This observation suggests that P-450arom belongs to a separate gene family which has been designated CYP1925. Aromatase catalyzes the conversion of androgens to estrone (E1), which is further converted to the potent estrogen estradiol (E2) by the enzyme 17β-HSD type 1 in the granulosa cell.
Development of Aromatase Inhibitors
Aromatase is a good target for selective inhibition because estrogen production is a terminal step in the biosynthetic sequence. There are two types of aromatase inhibitors; steroidal (type I inhibitors) and non-steroidal inhibitors (type II inhibitors). Type I steroidal aromatase inhibitors are all derivatives of androstenedione that act as a false substrate and bind irreversibly to the androgen-binding site with continuing treatment. For this reason, they are also called suicide inhibitors. 4-hydroxyandrostenedione, the first selective steroidal aromatase inhibitor to be used clinically, has proved to be effective in tamoxifen-resistant breast cancer patients and is available in many countries worldwide26. Type II non-steroidal aromatase inhibitors exert their function through binding to the heme moiety of the cytochrome P450 enzyme. The first of these inhibitors to be used clinically was aminoglutethimide, which induces a medical adrenalectomy by inhibiting many other enzymes involved in steroid biosynthesis27. Although aminoglutethimide is an effective hormonal agent in postmenopausal breast cancer, its use is complicated by the need for concurrent corticosteroid replacement, in addition to side effects like lethargy, rashes, nausea and fever that results in 8-15% of patients stopping treatment. The lack of specificity and unfavorable toxicity profile of aminoglutethimide have led to the search for more specific aromatase inhibitors. In addition, the above mentioned aromatase inhibitors were not able to completely inhibit aromatase activity in premenopausal patients28.
Aromatase inhibitors such as anastrozole, ZN 1033, (Arimidex®), Ietrozole, CGS 20267, (Femara®) and vorozole (Rivizor®) are selective AIs, available for clinical use in North America and other parts of the world for treatment of postmenopausal breast cancer. These triazole (antifungal) derivatives are reversible, competitive AIs, which are highly potent and selective (104, 106)29. Their intrinsic potency is considerably greater than that of aminoglutethimide, and at doses of 1-5 mg/day, inhibit estrogen levels by 97% to >99%. This level of aromatase inhibition results in estradiol concentrations below detection by most sensitive immunoassays. The high affinity of AIs for aromatase is thought to reside in the N-4 nitrogen of the triazole ring that coordinates with the heme iron atom of the aromatase enzyme complex. AIs are completely absorbed after oral administration with mean terminal t1/2 of approximately 50 hr (range, 30-60 hr). They are cleared from the systemic circulation mainly by the liver. Another Al available commercially is exemestane (Aromasin™).
In animal studies, letrozole resulted in increased FSH and LH when given to mature female rats and about a 30% increase in ovarian weight30. In the bonnet monkey, treatment with aromatase inhibitors to induce estradiol deficiency led to development of multiple normal Graafian follicles in vivo, and normal response of granulosa and theca cells to gonadotropins in vitro31. In vivo data describe a continuum of inhibition of aromatase, with aminoglutethimide (90%), vorozole (93%), anastrozole (97%), and letrozole (98.5%) displaying increasing potency and specificity32. Letrozole has an IC50 of 11.5 nM in vitro and ED50 of 1-3 μg/kg in vivo when given orally. The disposition of orally administered letrozole in healthy postmenopausal women is characterized by steady-state plasma concentrations in 4 to 8 hours, and a half-life of approximately 45 hours. The absolute systemic bioavailability of letrozole after oral administration was 100% compared with the same dose given intravenously33. Doses up to 30 mg have been well tolerated34. The lethal dose in mice and rats is approximately 2000 mg/kg. There is no experience in humans with an overdose of letrozole35.
The success of aromatase inhibition by letrozole in inducing ovulation in women with PCOS has been reported36. In a series of 10 patients with PCOS who either failed to ovulate (n=4) or ovulated with an endometrial thickness ≦5 mm (n=6) in response to CC administration, ovulation occurred in 7 of the 10 letrozole treated cycles (70%), with clinical pregnancy in 2 patients and chemical pregnancy in one patient. The mean number of mature follicles was 2.6, ranging from 1 to 4 follicles in the 7 ovulatory cycles. The mean level of estradiol on the day of hCG administration was 1076 pmol/L with mean estradiol per follicle of 378 pmol/L. This estradiol level allowed the growth of the endometrium to an adequate thickness that ranged from 0.7 cm to 0.9 cm on the day of hCG administration, showing the absence of antiestrogenic effects as seen with CC.
In a second study, comparable success of letrozole in inducing ovulation in 12 women with PCOS women, in addition to success in augmenting ovulation in a group of 10 ovulatory women. Patients in both groups tried CC in prior treatment cycles with an inadequate response. With letrozole treatment, ovulation occurred in 9 of 12 cycles (75%) and pregnancy was achieved in 3 patients (25%) in the PCOS group. In the ovulatory group, letrozole resulted in a mean number of 2.3 mature follicles and a mean endometrial thickness of 0.8 cm. Pregnancy was achieved in one patient (10%)37.
We have also studied the use of letrozole in conjunction with FSH for controlled ovarian super ovulation in both ovulatory women with unexplained infertility and anovulatory women with PCOS38. The use of letrozole was associated with a significantly lower FSH dose required for achievement of adequate ovarian super ovulation. The pregnancy rate and endometrial thickness with letrozole and FSH treatment was similar to FSH alone. We have also shown an improvement in ovarian response to FSH stimulation with the use of letrozole in low responders during ovarian stimulation39.
In U.S. Pat. No. 5,583,128 granted Dec. 10, 1996 to Bhatnagar, there is described the use of aromatase inhibitors for contraception in female primates of reproductive age without substantially affecting the menstrual cycle of the female primate. The contraceptive action of the aromatase inhibitors is reversible, that is to say once their use has been discontinued pregnancy can occur in the treated primates as early as the next cycle.
In U.S. Pat. No. 5,491,136 granted Feb. 13, 1996 to Peet et al, the use of aromatase inhibitors in the treatment of breast cancer is described.
The disclosures of all references referred to herein are incorporated herein by reference.